Human Immunodeficiency Virus (HIV) disease has killed over 12 million people world wide since it was first recognized in 1981. Today, it is estimated that over 31 million people are infected with HIV and nearly 16,000 new infections occur daily. The Untied Nations HIV/AIDS surveillance committee estimates that over 40 million people will be infected by the year 2000, the majority of these new infections will occur in developing countries. HIV disease, which includes acquired immune deficiency syndrome (AIDS), is caused by a virus belonging to the group Retroviridae (retroviruses). Specifically, HIV is a lentivirus which is a genus of the Retroviridae.
Many developing countries are confronted with health care issues such as malaria and tuberculosis (TB) which combine to kill more people annually than HIV. The World Health Organization can provide effective therapies against TB for 36 US dollars per person, and malaria can be treated for as little as $1.00 a month. However, even these seemingly insignificant amounts can cause severe economic hardship for individuals and families in developing countries. When the costs of these treatments are compared to the $12,000/individual/year cost associated with new combination drug therapies referred to as HAART (highly active anti-retroviral therapy), it becomes evident that HAART is an economic impossibility in developing countries where effective anti-retroviral therapy is needed most.
Combination drug therapy for HIV began to replace monotherapy (single drug treatments) in early 1996, and by 1999, the Food and Drug Administration (FDA) had approved 11 drugs which could be used in various HAART protocols. These eleven drugs are broken down into three classes which include nucleoside reverse-transcriptase inhibitors (NRTI) divided into two sub-groups A and B, non-nucleoside reverse-transcriptase inhibitors (NNRTI) and protease inhibitors (PI). Current recommendations for combination drug therapy include two NRTIs (one A and one B) combined with either a Pi or an NNRTI. This combination drug therapy has proven to be highly effective in significantly reducing viral load (the amount of HIV present in the blood or tissues of an infected person) and preventing the onset of severe immune deficiency in many compliant patients (patients who take their medications exactly as directed). However, there are significant drawbacks associated with combination drug therapy.
For many patients the toxic side effects diminish their quality of life to such an extent that they simply stop taking their medications. For others, the therapeutic schedules are so complicated and inconvenient that they find compliance nearly impossible to fit into a normal lifestyle. Still, many infected persons do not benefit from combination drug therapy due to virus strain variation and other unknown factors. Other patients experience excellent results initially, but due to mutations in the virus, they suffer viral load relapses in spite of full compliance with the therapeutic regime. Many of these patients can be treated with other drug combinations that knock the viral load back down, but the risk of mutation, repeated drug failure, and the onset of new drug side effects are persistent fears. In total, the side effects, psychological burden, cost and uncertainty of efficacy continue to take a steep toll on patients currently undergoing combination drug therapy, rendering the best available option for treating this deadly virus worse than the disease for many people.
Another significant limitation of combination drug therapy is that these treatment regimes do not completely eliminate the virus from the body. Due to the complex nature of our immune system and human retrovirus genetics, HIV is able to sequester itself inside dormant immune cells where it remains unaffected by the drugs. Consequently, if the patient stops taking the medications, a rapid resurgence in HIV viral load occurs, requiring the patent to take anti-retroviral drugs for life. Moreover, studies have demonstrated that seropositive (HIV infected) compliant patients who have undetectable virus in their body are still capable of transmitting the virus sexually or through contact with their blood. Ultimately, the best defense against any disease is prevention, and presently the best prophylaxis against the threats of infectious agents is vaccination.
Medical researchers have been seeking an effective HIV vaccine since the virus was first discovered in 1983. Previous vaccine efforts have included inactivated whole virus, structurally modified, inactivated whole virus, viral sub-unit vaccines including gag (group associated, or core antigens), pol (viral polymerases), and env (viral envelope antigens). In the latter group, both native and recombinant proteins have been investigated. Various vaccination techniques, including frequency of administration, routes of administration and adjuvant mixtures (inert ingredients mixed with the viral antigens to help stimulate the host response), have been tried. Many of these vaccine approaches have elicited detectable immune response in animals including humans, and a few have afforded the animal with limited protection against infection after being challenged with live virus. However, a safe and effective HIV prophylactic suitable for widespread human use remains elusive. Therefore, there is a pressing need for a cost effective, non-toxic, highly active treatment for HIV infected individuals, and even more importantly, for an effective prophylactic vaccine.
Recently, significant advances have been made in understanding the HIV disease process. For many years, researchers have been unable to explain the seemingly immediate and profound destruction of the immune system following the initial HIV infection. Equally puzzling was a phenomenon seen in a few patients referred to as long term non-progessors (LTNP). In LTNP patients, viral loads are high and the virus can be isolated easily from the HIV target immune cells such as CD4+ T lymphocytes (referred to herein as T4 cells). However, unlike the majority of infected individuals who develop AIDS, the LTNP do not demonstrate significant reduction in their T4 cells and do not progress to AIDS.
One possible, non-binding, theory that may explain these two phenomena involves a non-structural protein (a protein encoded by the virus genome that is not actually part of the virus itself) called trans-activator of transcription, or Tat for short. Tat is a variable RNA binding peptide of 86 to 110 amino acids in length that is encoded on two separate exons of the HIV genome. Tat is highly conserved among all human lentiviruses and is essential for viral replication. When lentivirus Tat binds to the TAR (trans-activation responsive) RNA region, transcription (conversion of viral RNA to DNA then to messenger RNA) levels increase significantly. The Tat protein associated with lentivirus virulence will be referred to hereinafter at C-Tat, or “conventional Tat.” Recently, it has been demonstrated that C-Tat increases viral RNA transcription and it has been proposed that C-Tat may initiate apoptosis (programmed cell death) in T4 cells and macrophages (a key part of the body's immune surveillance system for HIV infection) and possibly stimulates the over production of alpha interferon (αINF is a well established immunosuppressive cytokine). These, and other properties of lentivirus C-Tat proteins, have led to considerable scientific interest in C-Tat's role in pathogenesis and to the present inventor's proposal that Tat may act as a powerful immunosuppressant in vivo.
A potential key to lentivirus C-Tat pathogenesis may involve in its ability to trigger apoptosis. Conventional Tat initiates apoptosis by stimulating the expression of Fas Ligand (FasL) (a monomeric polypeptide cell surface marker associated with apoptosis) on the T4 cell and macrophage surface. When FasL is cross linked by binding with FAS (the counter part to FasL which is also expressed on a wide variety of cell types), the apoptotic system is activated. Consequently, the death of these essential T4 cells and macrophages is accelerated, resulting in extreme immunosuppression. Thus, extracellular C-Tat's presence early in the course of HIV infection could reduce a patient's immune response, giving the virus an advantage over the host. Furthermore, the direct destruction of T4 cells and induction of αINF production could help explain the lack of a robust cellular immune response seen in AIDS patients, as well as accounting for the initial profound immunosuppression.
Further support for this concept is found in a surprising new observation made by the present inventor who has demonstrated the Tat protein isolated from long term non-progressors is different from C-Tat found in AIDS patents. The Tat protein found in LTNP is capable of trans-activating viral RNA, however, LTNP Tat (designated herein after as IS-Tat for immuno-stimulatory Tat) does not induce apoptosis in T4 cells or macrophages and is not immunosuppressive. Moreover, T4 cells infected ex vivo with HIV isolated from LTNP (such cell lines are designated Tat TcL) can result in the over expression of IS-Tat proteins, often to the virtual exclusion of other viral proteins, that are strongly growth promoting rather than pro-apoptotic. The tat genes cloned from these Tat TcLs reveal sequence variations in two tat regions, at the amino terminus and within the first part of the second exon. These surprising discoveries could help explain why HIV infected LTNP T4 cells do not die off at the staggering rate seen in HIV infected individuals that progress to AIDS.
The present inventor has demonstrated that macrophages are approximately 1000 times more sensitive to Tat (both C-Tat and IS-Tat) than T4 cells which has led to the development of a new in vitro ultra-sensitive macrophage Tat bioassays which permit, for the first time, the direct in vitro measurement of C-Tat's immunosuppressive capacity. Using this new ultra-sensitive macrophage bioassay, the present inventor has been able to directly measure immunosuppressant activity of different Tats which aided the inventor in developing a new HIV vaccine strategy.
This new vaccine strategy uses non-immunosuppressive Tat (either IS-Tat or denatured C-Tat that has been rendered non-immunosuppressive), either alone or in combination with other HIV proteins, and with or without an adjuvant, to provide protection against HIV infection. This preparation can also be used as an immunotherapeutic in HIV infected individuals to prevent the profound immunosuppression associated with AIDS by inducing the inoculated person's immune system to produce neutralizing antibodies directed against indigenous C-Tat (Tat produced as a result of natural HIV infection).
The use of C-Tat protein as a potential HIV vaccine component has been previously described in the medical literature; however, these strategies have been either unsuccessful or impractical for human use. Previously published efforts required multiple inoculations over a protracted time period in order to induce an immune response. This may have been caused by the immunosuppressive activity of the C-Tat component of the vaccine itself. These previous investigators did not have a convenient and reliable in vitro assay system in which to assess their C-Tat preparations for immunosuppressive activity.
The safety testing conducted in these earlier studies looked for evidence of C-Tat toxicity in animals and trans-activation activity in cell culture, but not immunosuppressive activity directly. Some forms of C-Tat toxicity are not associated with C-Tat immunosuppression. Consequently, assays that merely measure non-immunosuppressive toxicity, such as short-term small animal models, cannot be reliably used to alert an investigator that his C-Tat vaccine preparation was immunosuppressive. As a result, if an immunosuppressive vaccine was unknowingly administered to a test animal, multiple vaccinations over a protracted time period would be required to induce an immune response.
It is yet another non-binding theory of the present inventor, that based on the observations with long-term CD4+ Tat T cell lines (Tat TcL), clinical observations, and experiments in animals, attenuated Tat (more specifically IS-Tat or, alternatively, C-Tat proteins that have been chemically or physically altered) may act as an immune stimulant activating T4 cells inducing their proliferation. This principle may help to explain the stable T4 levels seen in LTNP. Moreover, it is proposed that attenuated Tat may be useful as an adjuvant when co-administered with other active vaccine components such as, but not limited to, vaccines for other viruses, bacteria, rickettsia and cancer cells